The overall goals of this research proposal are to identify and characterize the insulin signal transduction pathways leading to functional regulation of the protein components directly involved in the intracellular trafficking, plasma membrane tethering, docking and fusion of GLUT4-containing vesicles. Substantial evidence has indicated that plasma membrane lipid raft microdomains provide an important spatially restricted compartment necessary for insulin biological action. These microdomains are self-organized into large rosette-like structures highly enriched in caveolin that serve as scaffolding or platform for the assembly of plasma membrane cortical actin (Cav-actin). Although it is well-established that dynamic actin remodeling plays an essential role in the GLUT4 translocation process the mechanism(s) responsible are completely unknown. Our preliminary data indicates that a certain subset of actin assembly proteins (beta1 integrin receptor subunit, talin and the type I phosphatidylinositol-4-phosphate 5-kinase) but not others, are co-localized to these Cav-Actin domains. In addition, GLUT4 vesicle fusion proteins (SNARE) have also been localized to these domains and appear to functionally interact with F-actin. To further examine the functional role of adipocyte lipid raft microdomains in mediating insulin signaling, we propose four specific aims, 1. We will reduce expression of the a1 integrin receptor subunit, talin and PI4P5 kinase by the use of siRNA in culture and by homologous recombination in vivo. In these experiments, we will assess the role of these proteins during the adipocyte differentiation process both in vivo and in vitro in terms of the assembly of actin and acquisition of insulin specific signaling processes. 2 We will assess the role of several SNARE proteins in GLUT4 vesicle docking versus fusion, effects on actin assembly and requirement for lipid raft microdomain localization. 3. We will reconstitute membrane vesicle fusion and determine the effect of these regulatory proteins on the in vitro fusion reaction. The effect of direct kinase phosphorylation and phosphorylation deficient mutants will be determined. As efforts to establish a plasma membrane fusion assay, we will assess the fusion reaction between these reconstituted membrane vesicles with adipocytes plasma membrane sheets. 4. We will use FRAP to assess the diffusion rates in isolated plasma membrane sheets and intact cells under a variety of conditions including disruption and/or stabilization of lipid rafts, cortical actin and microtubules. In a complimentary approach, we will also take advantage of the newly developed photoactivable GFP construct (PA-GFP) to examine diffusion from non-lipid raft and caveolin-enriched plasma membrane microdomains.